Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline and in accordance with GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
Not specified in the report.
Principles of method if other than guideline:
Guideline followed
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 7-Ethyl bicyclooxzaolidine (BIOBANTM CS-1246 Antimicrobial)
- Molecular formula (if other than submission substance): C7H13NO2
- Molecular weight (if other than submission substance): 143.2
- Physical state: Liquid
- Analytical purity: 96.85% ± 0.05%
- Impurities (identity and concentrations): Six impurities were present at concentrations higher than 0.1% area normalized.
- Lot/batch No.: PAO531LAN3

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Portage, Michigan)
- Age at study initiation: 8 weeks
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing:
During the study animals were housed singly in stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Cages had wire-mesh floors and were suspended above catch pans. Cages contained hanging feeder and a pressure activated nipple-type-watering system.
- Diet (e.g. ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form.
- Water (e.g. ad libitum): Municipal water was provided ad libitum.
- Acclimation period:
Upon arrival at the laboratory1, each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes. The animals were housed 2 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least seven days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0-22.1 °C
- Humidity (%): 48.7-50.5%.
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
None
Details on exposure:
Dose Range-Finding Test:
This assay was conducted to aid the selection of dose levels for the micronucleus test. Groups of mice (4/sex/dose) were treated with 0, 500, 1000, and 2000 mg/kg/day of the test material on two consecutive days. Observations were made 72 hours after dosing or until Day 4 of the study for any clinical signs of toxicity. Day 1 was defined as the first day of test material administration. Animals were dosed on January 10 and 11, 2002.

Micronucleus Test:
Groups of male mice were administered 0, 500, 1000, or 2000 mg/kg/day of the test material on two consecutive days. Animals were dosed on January 29 and 30, 2002. CP was administered only once at a dose level of 120 mg/kg BW on January 30, 2002. The dose levels were based on the results of the range finding test. There were 6 mice/dose except in the 2000 mg/kg/day group where an additional group of two mice were dosed
as possible replacements in the event of deaths occurring among the treated animals of this group. The mice were observed daily and signs of toxicity or deaths occurring during the observation period were recorded. Approximately 24 hours after the last dosing, bone marrow samples were collected from all animals.
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
Groups of male mice were administered 0, 500, 1000, or 2000 mg/kg/day of the test material on two consecutive days. Animals were dosed on January 29 and 30, 2002. CP was administered only once at a dose level of 120 mg/kg BW on January 30, 2002.
Post exposure period:
Dose Range-Finding Test:
Observations were made 72 hours after dosing or until Day 4 of the study for any clinical signs of toxicity.

Micronucleus Test: 24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, and 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Dose Range-Finding Test: 4/sex/group
Micronucleus Test: 6/group
Control animals:
yes
Positive control(s):
cyclophosphamide-monohydrate (CP)

- Route of administration: Oral gavage
- Doses / concentrations: Positive control (CP) was administered once, approximately 24 hr. (± 1 hr.) before sacrifice using the same method as the test material and negative control.

Examinations

Tissues and cell types examined:
Bone marrow samples with PCE
Details of tissue and slide preparation:
Bone Marrow Sampling:
At the end of the specified interval following treatment, the animals were euthanized using central nervous system depression following exposure to carbon dioxide in a closed chamber. Bone marrow samples were obtained from both femurs from 6 mice/dose level as described below. Bone marrow samples were collected from only one mouse in the additional group of mice at the 2000 dose level since there was one death prior to necropsy among the main group of this dose. The distal end of the femur was severed to expose the marrow cavity after separating the bone from the adjoining muscles. A 25-gauge needle was used to aspirate the bone marrow into a 3-ml disposable plastic syringe containing approximately 1.0 ml of fetal bovine serum (GIBCO, Grand Island, New York). After aspiration, the contents of the syringe were transferred into a 1.5-ml centrifuge tube containing 1.0 ml of serum. The cells were resuspended in the serum by gentle aspiration using the syringe and needle. The tubes were centrifuged at 1000 rpm (approximately 80 g) for approximately 5 minutes in a tabletop centrifuge. The supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipette. Wedge smears were prepared on microscope slides using small portions of the cell suspension. The slides were allowed to air dry and stained with Wright-Giemsa using an automatic slide stainer, (Ames Hema-Tek, Miles Scientific, Naperville, Illinois).

Slide Scoring:
All slides were coded, scored, and decoded upon completion to control for bias. Two thousand PCE were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with smooth contours and varying shapes such as round, almond, or ring (Schmid, 1976). The ratio of PCE to NCE in the bone marrow was determined in the micronucleus test by examining 200 erythrocytes. The ratio was expressed as PCE X 100/PCE+NCE.
Evaluation criteria:
A test was considered valid if all of the following conditions were met:
• The range of MN-PCE values in the negative controls was within reasonable limits of the recent (past five years) laboratory background range.
• There was a significant increase in the incidence of MN-PCE in the positive control treatment as compared to the concurrent negative controls.
• The mean percent PCE was greater than 5% in one or more of the test material treated groups.

A test material was considered positive in this assay if the following was met:
• Statistically significant increase in MN-PCE frequency at one or more dose levels accompanied by a dose response.

A test material was considered negative in this assay if all of the following criteria were met:
• No statistically significant dose related increase in MN-PCE was compared to the negative control.
• The MN-PCE values in the treated animals were within reasonable limits of the recent (within five years) laboratory historical background range.
• The mean percent PCE values in one or more of the test material treated groups was
greater than 5%.

A test result not meeting the criteria for either for the positive or the negative response was considered to be equivocal.
Statistics:
Statistics
The raw data on the counts of MN-PCE for each animal were first transformed by adding one to each count and then taking the natural log of the adjusted number. The transformed MN-PCE data and the data on percent PCE were analyzed separately by a one-way analysis of variance (Winer, 1971). Pairwise comparisons of treated vs. control groups were done, if the dose effect was significant, by Dunnett's t-test, one-sided (upper) for MNPCE and two-sided for the percent PCE (Winer 1971). Linear dose-related trend tests were performed only if any of the pairwise comparisons yielded significant differences. The alpha level at which all tests were conducted was 0.05.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Dose Range-Finding Test:
Targeted dose levels of 0, 500, 1000 and 2000 mg/kgBW BIOBAN™ CS-1246 were used in the range-finding portion of the assay. The actual doses ranged from 99 to 103% of the targeted values as estimated by the analytical methods (Table 1).
There were no appreciable changes in the body weights of mice (Summary data, Tables 2 and 3; Individual data Tables 4 and 5). Minimal signs of toxicity were observed among the highest dose level during the study. One female dosed with 2000 mg/kg exhibited decreased activity and was cold to the touch. A second female and one male at the 2000 mg/kg dose level had perioral soiling. A single death attributed to treatment with BIOBAN™ CS-1246 occurred in the male 2000 mg/kg dose group and one female in the 1000 mg/kg dose level died as a result of a gavage error (Summary data, Table 6; Individual data, Tables 7 and 8). No clinical signs of toxicity were observed in mice receiving 500 or 1000 mg/kgBW of the test article. One high-dose male mouse exhibited a significant decrease in body temperature that was attributed to the moribund status of the animal. There were no remarkable treatment-related changes in the body temperature of remaining mice following treatment with the test material (Summary data, Tables 9 and 10; Individual data, Tables 11 and 12).

Micronucleus Test:
Based upon the results of the range finding study only males were evaluated in the main study, since no significant differences in response to the test article were observed between male and female mice. The limit dose of 2000 mg/kg/day BIOBAN™ CS-1246 was selected as the high-dose. Selection of the limit dose was based upon the results of the range finding test where one male mouse receiving 2000 mg/kg died prior to the end of the observation period and minimal signs of toxicity were observed in the remaining mice of both sexes. The middle- and low-doses were 1000 mg/kgBW and 500 mg/kgBW, respectively. The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 97 to 103% of the targeted values (Table 13).
The treatments did not have a remarkable effect on the body weight of the animals (Summary data, Table 14; Individual Table 15). Three of eight high-dose mice died prior to the end of the study. Mice that died spontaneously appeared incoordinated and were minimally active. One of the three spontaneous mice exhibited convulsions prior to death. A single spontaneous death occurred at the 1000 mg/kg/day group immediately after administration of the first dose as the result of a gavage error. In order to maintain an N of six for this dose group, a spare animal was subsequently dosed as a replacement. No remarkable observations were noted in surviving mice dosed with 2000 mg/kg/day or in mice dosed with 1000, or 500 mg/kg/day BIOBAN™ CS-1246 (Summary data, Table 16; Individual data, Table 17).
A summary of the data on the frequencies of MN-PCE and percent PCE observed in various treatment groups of male mice is presented in Table 18 and the laboratory historical control data are shown in Table 20. The individual animal data are presented in Table 19. There were no significant differences in MN-PCE frequencies between the groups treated with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated polychromatic erythrocytes in the positive control group (Table 18).
The percent PCE values observed in the test material-treated animals were not significantly different from the negative control values (Table 18). The percent PCE values of the positive control animals were slightly lower, but not statistically significant, than those of the negative control animals

Any other information on results incl. tables

Refer attachmet: 1 (Includes the below mentioned TABLES)

Table 2. Summary Body Weights (G) – Males (Range Finding Test)

Table 3. Summary Body Weights (G) – Females (Range Finding Test)

Table 4. Individual Body Weights (G) – Males (Range Finding Test)

Table 5. Individual Body Weights (G) – Females (Range Finding Test)

Table 6. Summary In-Life Observations – Males/Females (Range Finding Test)

Table 7. Individual Animal Observations – Males (Range Finding Test)

Table 8. Individual Animal Observations – Females (Range Finding Test)

Table 9. Summary Animal Body Temperature Data – Males (Range Finding Test)

Table 10. Summary Animal Body Temperature Data – Females (Range Finding Test)

Table 11. Individual Temperature Reporting – Males (Range Finding Test)

Table 12. Individual Temperature Reporting – Females (Range Finding Test)

Table 14. Summary Body Weights (G) – Males (Micronucleus Test)

Table 15. Individual Body Weights (G) – Males (Micronucleus Test)

Table 16. Summary In-Life Observations – Males (Micronucleus Test)

Table 17. Individual Animal Observations – Males (Micronucleus Test)

Table 18. Summary Micronucleated Polychromatic Erythrocytes (MNPCE) Frequencies and % Polychromatic Erythrocytes (%PCE) – Males

Table 19. Individual MNPCE and % PCE – Males (Micronucleus Test)

Table 20. Recent Historical Control Data

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions used, BIOBAN™ CS-1246 was considered to be negative in the mouse bone marrow micronucleus test at dose levels up to the limit dose of 2000 mg/kg/day.
Executive summary:

The in vivo genotoxic potential of BIOBAN™ CS-1246 was evaluated by examining the incidence of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow. The test material was administered to male CD-1 mice by single oral gavage on two consecutive days at dose levels of 0 (negative control), 500, 1000, and 2000 mg/kg body weight (BW). The highest dose level of 2000 mg/kgBW was the limit dose and was based upon the results of a range-finding test that demonstrated moderate toxicity at the highest dose level with no significant difference in the toxicological response to treatment between male and female mice. The concentrations of the test material in the dosing solutions were verified by analytical methods. Groups of animals, 6/sex/dose, were sacrificed at 24 hours after the second treatment for the collection of femoral bone marrow and evaluation of PCE (2000/animal) for MN. The proportion of PCE among erythrocytes was determined based upon 200 erythrocytes per animal and the results expressed as a percentage. Mice treated with 120 mg/kgBW cyclophosphamide monohydrate and sacrificed 24 hours later served as positive controls. Clinical signs of toxicity were noted only in the 2000 mg/kg/day dose group where three mice died prior to the scheduled sacrifice. One mouse in the 1000 mg/kg/day group died due to a gavage error on the first day of dosing and was subsequently replaced with a spare animal. There were no statistically significant increases in the frequencies of MN-PCE in groups treated with the test material as compared to the negative controls. The positive control animals showed a significant increase in the frequency of MN-PCE as compared to the negative control animals. Under the experimental conditions used, BIOBAN™ CS-1246 was considered to be negative in the mouse bone marrow micronucleus test at dose levels up to the limit dose of 2000 mg/kg/day.